Photolabeling of CheR methyltransferase with S-adenosyl-L-methionine (AdoMet). Studies on the AdoMet binding site.

CheR methyltransferase from Salmonella typhimurium was directly photolabeled with S-adenosyl-L-[methyl-3H]methionine. The labeled protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then was detected by fluorography. The methylase-S-adenosyl-L-methionine adduct was found to be stable under the experimental conditions employed. Labeling was found to be a function of the concentration of enzyme, S-adenosyl-L-methionine (AdoMet), and the intensity and time of UV irradiation. The extent of labeling and protein methylation was found to be inhibited by S-adenosyl-L-homocysteine, S-adenosyl-L-ethionine, and sinefungin, which are known to compete with AdoMet for the same binding site on the enzyme. Our earlier data showed that the enzyme has 2 cysteine residues and that these are important for enzyme activity. Here, we show that sulfhydryl reagents inhibit the photolabeling of the substrate to the enzyme, indicating the presence of cysteine in the vicinity of the substrate-binding site. We also found that when Cys31 was modified to Ser, no photolabeling of CheR was observed, whereas a modification of Cys229 to Ser had little effect on the ability of AdoMet to label the enzyme. This suggests that Cys31 is located at or near AdoMet-binding site. The labeled protein was cleaved at tryptophan residues, generating two major fragments, each containing 1 cysteine residue. SDS-PAGE and fluorography of the cleaved products indicated the presence of the label being associated with the Cys31 fragment. Similar results were obtained when the labeled protein was cleaved at glutamic acid residues using V8 protease. A tryptic digest of the labeled protein showed two radioactive peptide peaks when subjected to separation on reverse phase high pressure liquid chromatography. The labeled peptides were further digested to free amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that Cys31 may be involved with substrate binding, as well as with catalysis.

Probing the role of cysteine residues in the CheR methyltransferase.

The CheR methyltransferase catalyzes the transfer of methyl groups from S-adenosylmethionine to specific glutamyl residues in bacterial chemoreceptor proteins. Studies with sulfhydryl reagents such as p-chloromercuribenzoate, N-ethylmaleimide, and 5,5'-dithiobis(2-nitrobenzoate) suggest that a cysteine residue is required for enzyme activity. The nucleotide sequence of the cheR gene predicts a 288-amino acid protein with cysteine residues at positions 31 and 229. To ascertain the role of these cysteine residues in the structure and function of the enzyme, oligonucleotide-directed mutagenesis was used to change each cysteine to serine. Whereas the Cys229-Ser mutation had essentially no effect on transferase activity, the Cys31-Ser mutation caused an 80% decrease in enzyme activity. The double mutant in which both cysteines were replaced by serines also had markedly reduced transferase activity. Preincubation of the wild type or Cys229-Ser proteins with either S-adenosylmethionine or beta-mercaptoethanol protected it from inhibition by sulfhydryl reagents, whereas prior incubation with the second substrate, the Tar receptor, gave partial protection. From these studies, Cys31 appears to be necessary for enzyme activity, and it seems to be located in the vicinity of the active site.

The kinetic mechanism of S-adenosyl-L-methionine: glutamylmethyltransferase from Salmonella typhimurium.

The kinetic mechanism of the CheR methyltransferase, S-adenosyl-L-methionine (AdoMet): protein-L-glutamate O-methyltransferase (EC 2.1.1.24), from Salmonella typhimurium was investigated. Initial velocity, product inhibition, and binding studies were performed, and from the data obtained, it was determined that the mechanism of the reaction catalyzed by the enzyme is random. Initial velocity rates were measured with varied amounts of both substrates, and double-reciprocal plots gave patterns which converged on or near the abscissa. The products, S-adenosyl-L-homocysteine and methylated receptor, were found to be competitive inhibitors with respect to both AdoMet and receptor. Equilibrium dialysis and immunoprecipitation studies indicated that the two substrates can bind to the enzyme independent of each other. These results are consistent with a random mechanism with no abortive complexes being formed. The Michaelis constants calculated for AdoMet and receptor were 8.62 microM and 2.03 mg/ml total membrane protein (approximately 2.10 microM Tar protein), and the apparent dissociation constants of AdoMet and the receptor were 16.8 microM and 4.07 mg/ml total membrane protein (approximately 4.2 microM Tar protein), respectively. The Kd of AdoMet for the enzyme was 10.9 microM as determined by binding studies.

Purification and characterization of the S-adenosylmethionine:glutamyl methyltransferase that modifies membrane chemoreceptor proteins in bacteria.

The enzyme (EC 2.1.1.24) from Salmonella typhimurium that catalyzes the S-adenosylmethionine-dependent methyl esterification of glutamyl residues in membrane chemoreceptor proteins has been purified to homogeneity, and the nucleotide sequence of the gene coding for this protein, cheR, has been determined. The molecular weight, amino acid composition, and N-terminal amino acid sequence of the purified protein correspond to the values predicted from the sequence of the gene. The pure protein is a 33-kDa monomer. Kinetic studies indicate that, at levels of receptor and S-adenosylmethionine present in wild type cells, the transferase is nearly saturated. The enzyme has a relatively low turnover number, approximately 10 mol of methylester formed per mol of enzyme per min; and there appear to be only approximately 200 methyltransferase monomers per wild type cell.

Active site of the enzyme which demethylates receptors during bacterial chemotaxis.

The CheB methylesterase catalyzes the hydrolysis of glutamyl methyl esters in bacterial chemoreceptor proteins. Studies with residue-specific inhibitors suggest that a cysteine residue is required. The nucleotide sequence of the cheB gene predicts a 349-amino acid protein with cysteine residues at positions 207 and 309. Oligonucleotide-directed mutagenesis was used to change each cysteine to an alanine. Whereas the Cys207-Ala mutation had essentially no effect on esterase activity, the Cys309-Ala mutation caused a complete inactivation of the enzyme. Cys309 is located adjacent to a sequence of amino acids which is characteristic of the beta-alpha-beta motif found in a number of nucleotide binding proteins associated with receptor function in vertebrate tissues. A central feature of this structure is Gly-X-Gly-X-X-Gly. Mutation of the second glycine in this region (Gly284) to a valine also caused a complete loss of esterase activity.

Multiple forms of the CheB methylesterase in bacterial chemosensing.

The methylesterase which catalyzes demethylation of chemotactic membrane receptors in Salmonella typhimurium has been purified and characterized. Two forms of the enzyme have been isolated from cell extracts. One corresponds in molecular weight, Mr = 37,000, and amino acid composition to the predicted product of the structural gene for the methylesterase, cheB. The other is a proteolytic fragment, Mr = 21,000, corresponding to the C-terminal three-fifths of the intact CheB protein. The specific activity of the 21-kDa enzyme is at least 15-fold greater than that of its 37-kDa precursor. We conclude that the CheB protein is composed of at least two structurally distinct portions: a C-terminal catalytic domain, and an N-terminal region which modulates esterase activity.

Purification and characterization of succinyl-CoA: tetrahydrodipicolinate N-succinyltransferase from Escherichia coli.

Tetrahydrodipicolinate succinylase, an enzyme involved in the diaminopimelate-lysine pathway, was purified 1900-fold from crude extracts of Escherichia coli. The enzyme catalyzes the formation of CoA and N-succinyl-2-amino-6-keto-L-pimelate from succinyl-CoA and tetrahydrodipicolinate. The purified enzyme was shown to be homogeneous by polyacrylamide gel electrophoresis. The Stokes radius of the enzyme was determined from its elution volume on a Sephacryl S300 column and its sedimentation constant from sucrose density gradient centrifugation. These were 35 A and 4.7 (S20,w), respectively. The enzyme consists of two subunits each with a mass of 31,000 daltons, as determined using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Tetrahydrodipicolinate succinylase was shown to be a sulfhydryl enzyme. It has a pH optimum of 8.2. The equilibrium lies predominantly in favor of product formation but the reverse reaction can be demonstrated in vitro.